Tumor-associated chronic inflammation has long been linked to both hematogenous and lymphatic metastases, both of which directly correlated with reduced cancer patient survival. Beyond this correlation, little is known about the molecular basis of inflammation-associated metastasis. Studies designed to interrogate this question could advance development of effective therapies to improve patient outcomes. The proposed investigation will provide molecular insight into central mechanisms of inflammation that promote metastasis. The main pathway controlling inflammatory responses, Nuclear Factor kappa B (NF-?B), is frequently hyperactive in epithelial tumors due to over-production of inflammatory cytokines by neoplastic cells as well as by host cells within the tumor microenvironment. Our preliminary studies demonstrate that activation of the NF-?B pathway increases expression of vascular endothelial growth factor receptor-3 (VEGFR-3), the main receptor driving lymphangiogenesis and lymphatic metastasis. Evidence is also presented that in lymphatic endothelial cells (LECs) VEGFR-3 expression might be regulated by both the p50 subunit of NF-?B and the lymphatic-specific transcription factor, Prox1. Consistent with hyperactive NF-?B signaling, we found that NF-?B-inducing factors IL-1, MIF and KC/CXCL1 are overexpressed in pro-lymphangiogenic breast tumor lines and act in concert with a VEGFR-3 specific ligand, VEGF-C156S, to synergistically induce LECs proliferation. Collectively, our findings imply that NF-?B and Prox1 activate the VEGFR-3 promoter, likely leading to increased VEGFR-3 expression and higher receptor density on the surface of LECs. Because the density of VEGFR-3 receptors is a likely rate-limiting step during lymphangiogenesis, we hypothesize that NF-?B and Prox1 mediated increase of VEGFR-3 transcription is crucial for induction of tumor lymphangiogenesis. To test this hypothesis, we propose the following Specific Aims: (1) Delineate the effects of NF-?B dependent inflammatory mediators IL-1, MIF and KC/CXCL1 on VEGFR-3 expression, activation of VEGFR-3 signaling and LEC stimulation in vitro; (2) Delineate the role of Prox1 in NF-?B-mediated regulation of VEGFR-3 expression; (3) Define the role of host and tumor-derived IL-1, MIF and KC/CXCL1 cytokines in induction of breast cancer-associated lymphangiogenesis and lymphatic metastasis in vivo. Successful completion of these aims is anticipated to provide the molecular basis imperative for the design of therapies specifically targeting tumor lymphangiogenesis and metastasis, thus advancing our overall goal of improving cancer patient survival. PUBLIC HEALTH RELEVANCE: Epithelial malignancies frequently have an inflammatory component that has long been linked to increased vessel formation and incidence of metastasis. We propose to delineate the molecular events that associate tumor inflammation, the formation of new lymphatic vessels and lymphatic metastasis. This information will help to identify novel targets for inhibition of lymphatic metastasis. Because metastasis is the primary cause of mortality from cancer, these studies have the potential to significantly improve health outcomes in a large number of cancer patients.